The Na+/Ca2+ exchanges if the principal Ca2+ extrusion mechanism in cardiomyocytes and plays an important role in [Ca2+]i regulation in other cell types. While the amino and carboxy ends of the protein contain the machinery necessary for ion transport, domains of the protein critical for its regulation are only now being defined. The central intracellular "loop region" has been shown to regulate function through three domains: 1) exchanges inhibitory peptide (XIP), 2) Na+ binding, 3) Ca2+ binding. Recently, the PI identified a fourth region of the intracellular loop that plays an important regulatory role (see preliminary results). This region defines the tissue-specific isoforms and arises as six exons (A, B, C, D, E and F) of the NCX1 gene are alternatively spliced (AS). The AS region appears to underlie the differences in behavior of the Na+/Ca2+ exchanges when function is examined in native cells or in expression systems. The proposed experiments extend preliminary observations that demonstrate functional differences between alternatively spliced isoforms of the Na+/CA2+ exchanger. The questions include: 1) How do distinct isoforms of the Na+/Ca2+ exchanger respond differently to the established intracellular regulators: [Ca2+]i, [Na+]i and pHi? 2) How does activation of protein kinase A (PKA) affect the Na+/Ca2+ exchanger? What are the effects of the exchanger after activation of PKA? 3) How does membrane potential affect the alternatively spliced isoforms? The proposed work uses the whole-cell patch clamp technique in HEK293 expression system in combination with other alternative methods which include giant exised patch clamp, two-electrode voltage clamp and 45Ca2+ flux methods to study the function of isoforms of the Na+/Ca2+ exchanger. An external epitope tag on the Na+/Ca2+ exchanger permits the quantitation of expressed protein in cells. New methods to enrich for cells expressing of the Na+/Ca2+ exchanger and to quantitatively study its function will be used. Other state-of-the-art tools will be employed including confocal microscopy, expression of NCX1 isoforms using adenovirus systems, fluorescence measurements of intracellular substances and other diverse molecular methods. The proposed work will investigate, for the first time, the relationship between tissue specific isoforms of the Na+/Ca2+ exchanger and its function. The PI's approach will lay the foundation for continued investigation of links between structure and function of this and other transport proteins. The work is part of the PI's long term plans to understand Ca2+ signaling in biology.